1997 Fiscal Year Annual Research Report

核内蛋白との相互作用によるDNAポリメラーゼα・プライマーゼの複製開始制御

Research Project

Project/Area Number	07457037
Research Institution	Nagoya University
Principal Investigator	吉田松年名古屋大学, 医学部, 教授 (70090420)
Co-Investigator(Kenkyū-buntansha)	鈴木元名古屋大学, 医学部, 講師 (80236017) 小泉恵子名古屋大学, 医学部, 助教授 (00118027)
Keywords	網膜芽細胞腫遺伝子Rb / DNAポリメラーゼα / バキュロウィルス / cdk / cyclin / 複製因子 / RP-A
Research Abstract	哺乳類細胞のDNA複製酵素と調節因子との蛋白-蛋白相互作用を解析してG1/S移行の機構にアプローチすることを目的とする。 Rb蛋白はG1からS期に進むとcdk蛋白リン酸化酵素により高リン酸化型に変換する。この高リン酸化型Rb蛋白は従来不活性であると考えられていたが、我々は高リン酸化RbがS期を通して存続することに注目し、これがS期の進行に未知の役割を果たしていると考え、精製Rb蛋白の複製酵素に対する影響をin vitroにて検討した。バキュロウイルス昆虫細胞系で発現させ抗Rb抗体カラムで精製したRb蛋白はDNAポリメラーゼαの活性を促進した。昆虫細胞で発現されたRb蛋白は中程度のリン酸化型であり、これを脱リン酸化酵素PP2Aで処理すると促進活性は低下し、逆に、cdk2/cyclineEを共発現して高度リン酸型にすると促進は増強した(Takemura et al.Oncogene(1997)15,2483-2492)。この結果は、Rb蛋白はG1期には主としてE2Fなどの転写因子を不活性化する事によって複製系酵素蛋白の発現を阻害し、DNA複製の開始をブロックしているが、cdk/cyclin系によりリン酸化を受けるとE2Fに対する結合能を喪失して転写制御が解消すると同時に、リン酸化Rbそれ自体が複製酵素DNAポリメラーゼαを促進して、複製反応に促進的に作用するS phase prompterの一つ事であるを示唆する。我々は抗DNAポリメラーゼα・モノクローナル抗体を用いた免疫組織化学的解析から、核内ではDNAポリメラーゼα・プライマーゼ複合体は大きな構造体としてDNA複製部位に局在し、細胞周期に伴い離合集散を繰り返す事を明らかにした。この複製複合体の実体に迫るため、平成7年度及び8年度においてはDNAポリメラーゼαと蛋白相互作用によって結合する核内蛋白を検索した結果、T因子、MAPII、ポリADPリボシル化酵素(PARP)等を同定した。またDNAポリメラーゼα・プライマーゼ複合体に結合するDNAヘリカーゼ、α酵素の複製忠実度を向上させ、又プライマーゼの反応開始特異性を変えるRP-A(HSSB)もRF-C、PCNAとならんで複製複合体の構成因子であることが示唆された。

Research Products

(21 results)

All Other

All Publications (21 results)

[Publications] SHOJI-KAWAGUCHI Mami: "Selective Inhibition of DNA Polymerase ε by Phosphatidylinostitol." J.Biochem.117. 1095-1099 (1995)
[Publications] MURATE Takashi: "Herbimycin A Inhibits Phorbol Ester-Induced Morphologic Changes,Adhesion,and Megakaryocytic Differentiation of the Leukemia Cell Line,MEG-01." Proc.Soc.Exp.Biol.Med.209. 270-278 (1995)
[Publications] KOBAYASHI Jun'ichi: "Flavocristamides A and B,New DNA Polymerase α Inhibitors from a Marine Bacterium Flavobacterium sp." Tetrahedron. 51. 10487-10490 (1995)
[Publications] MURAKAMI-MUROFUSHI Kimiko: "Selective Inhibition of DNA Polymerase-α Family with Chemically Synthesized Derivatives of PHYLPA,A Unique Physarum Lysophosphatidic Acid." Biochim.Biophys.Acta. 1258. 57-60 (1995)
[Publications] MIZUNO Shin-ichi: "Portal Vein Branch Occlusion Induces Cell Proliferation of Cholestatic Rat Liver." J.Surg.Res.60. 249-257 (1996)
[Publications] IZUTA Shunji: "Chain Termination with Sugar-modified Nucleotide Analogs in the DNA Synthesis by DNA Polymerase γ." Nucleosides and Nucleotides. 15. 683-692 (1996)
[Publications] MIZUSHINA Yoshiyuki: "Fatty Acids Selectively Inhibit Eukaryotic DNA Polymerase Activities in Vitro." Biochim.Biophys.Acta. 1308. 256-262 (1996)
[Publications] OISHI Takashi: "Purification and Characterization of Nuclear Alksline Phospholipase A2 in Rat Ascites Hepatoma Cells." FEBS Lett.394. 55-60 (1996)
[Publications] SUZUKI,Motoshi: "Replication Protein-A Mediates the Association of Calf Thymus DNA Polymerase α-DNA Primase Complex with Guanine-Rich DNA Sequence." J.Biochem.120. 766-772 (1996)
[Publications] SAVOYSKY Ericka: "Down-regulation of Telomerase Activity is an Early Event in the Differentiation of HL60 Cells." Biochem.Biophys.Res.Commun.226. 329-334 (1996)
[Publications] TAKEUCHI Eiji: "Ligation of Portal Vein Branch Induces DNA Polymerase α,δ,and ε in Nonligated Lobes." J.Surg.Res.65. 15-24 (1996)
[Publications] MIZUSHINA,Yoshiyuki: "Screening of Inhibitor of Eukaryotic DNA polymerases Produced by Microorganisms." The Journal of Antibiotics. 49. 491-492 (1996)
[Publications] BANNO Yoshiko: "Nuclear ADP-ribosylation Factor (ARF)-and Oleate-dependent Phospholipase D(PLD)in Rat Liver Cells." J.Biol.Chem.272. 5208-5213 (1997)
[Publications] TAMIYA-KOIZUMI Keiko: "Inhibition of DNA Primase by Sphingosine and lts Analogues Parallels with Their Growth Suppression of Cultured Human Leukemic Cells." Bichem.Mol.Biol.Int.41. 1179-1189 (1997)
[Publications] TAKEUCHI Eiji: "Human Hepatocyte Growth Factor in Bile : An Indicator of Posthepatechtomy Liver Function in Patients With Biliary Tract Carcinoma." Hepatology. 26. 1092-1099 (1997)
[Publications] TAKEMURA Masaharu: "Phosphorylated Retinoblastoma Protein Stimulates DNA Polymerase α." Oncogene. 15. 2483-2492 (1997)
[Publications] MIZUSHINA Yoshiyuki: "The Inhibitory Action of Fatty Acids on DNA Polymerase β." Biochim.Biophys.Acta. 1336. 509-521 (1997)
[Publications] TAKEUCHI Eiji: "Research review : DNA polymerases as molecular markers of the regenerating capacity of hepatocytes." J.Hep.Bil.Pancer Surg.4. 254-262 (1997)
[Publications] HORIE Tadashi: "A5'-monophosphate Form of Bredinin Selectively Inhibits the Activities of Mammalian DNA Polymerases In Vitro." Int.J.Mol.Med.1. 83-90 (1998)
[Publications] KATO,Osamu: "Serum DNA Polymerase β as an Indicator for Fatal Liver lnjury of Rat Induced by D-Galactosamine Hydrochloride and Lipopolysaccharide." Biochim.Biophys.Acta. (in press).
[Publications] 吉田松年: "DNA複製・修復と発癌(松彰昭夫編)" 羊土社, (1996)

1997 Fiscal Year Annual Research Report

核内蛋白との相互作用によるDNAポリメラーゼα・プライマーゼの複製開始制御

Principal Investigator

吉田 松年 名古屋大学, 医学部, 教授 (70090420)

Research Products

[Publications] SHOJI-KAWAGUCHI Mami: "Selective Inhibition of DNA Polymerase ε by Phosphatidylinostitol." J.Biochem.117. 1095-1099 (1995)

[Publications] MURATE Takashi: "Herbimycin A Inhibits Phorbol Ester-Induced Morphologic Changes,Adhesion,and Megakaryocytic Differentiation of the Leukemia Cell Line,MEG-01." Proc.Soc.Exp.Biol.Med.209. 270-278 (1995)

[Publications] KOBAYASHI Jun'ichi: "Flavocristamides A and B,New DNA Polymerase α Inhibitors from a Marine Bacterium Flavobacterium sp." Tetrahedron. 51. 10487-10490 (1995)

[Publications] MURAKAMI-MUROFUSHI Kimiko: "Selective Inhibition of DNA Polymerase-α Family with Chemically Synthesized Derivatives of PHYLPA,A Unique Physarum Lysophosphatidic Acid." Biochim.Biophys.Acta. 1258. 57-60 (1995)

[Publications] MIZUNO Shin-ichi: "Portal Vein Branch Occlusion Induces Cell Proliferation of Cholestatic Rat Liver." J.Surg.Res.60. 249-257 (1996)

[Publications] IZUTA Shunji: "Chain Termination with Sugar-modified Nucleotide Analogs in the DNA Synthesis by DNA Polymerase γ." Nucleosides and Nucleotides. 15. 683-692 (1996)

[Publications] MIZUSHINA Yoshiyuki: "Fatty Acids Selectively Inhibit Eukaryotic DNA Polymerase Activities in Vitro." Biochim.Biophys.Acta. 1308. 256-262 (1996)

[Publications] OISHI Takashi: "Purification and Characterization of Nuclear Alksline Phospholipase A2 in Rat Ascites Hepatoma Cells." FEBS Lett.394. 55-60 (1996)

[Publications] SUZUKI,Motoshi: "Replication Protein-A Mediates the Association of Calf Thymus DNA Polymerase α-DNA Primase Complex with Guanine-Rich DNA Sequence." J.Biochem.120. 766-772 (1996)

[Publications] SAVOYSKY Ericka: "Down-regulation of Telomerase Activity is an Early Event in the Differentiation of HL60 Cells." Biochem.Biophys.Res.Commun.226. 329-334 (1996)

[Publications] TAKEUCHI Eiji: "Ligation of Portal Vein Branch Induces DNA Polymerase α,δ,and ε in Nonligated Lobes." J.Surg.Res.65. 15-24 (1996)

[Publications] MIZUSHINA,Yoshiyuki: "Screening of Inhibitor of Eukaryotic DNA polymerases Produced by Microorganisms." The Journal of Antibiotics. 49. 491-492 (1996)

[Publications] BANNO Yoshiko: "Nuclear ADP-ribosylation Factor (ARF)-and Oleate-dependent Phospholipase D(PLD)in Rat Liver Cells." J.Biol.Chem.272. 5208-5213 (1997)

[Publications] TAMIYA-KOIZUMI Keiko: "Inhibition of DNA Primase by Sphingosine and lts Analogues Parallels with Their Growth Suppression of Cultured Human Leukemic Cells." Bichem.Mol.Biol.Int.41. 1179-1189 (1997)

[Publications] TAKEUCHI Eiji: "Human Hepatocyte Growth Factor in Bile : An Indicator of Posthepatechtomy Liver Function in Patients With Biliary Tract Carcinoma." Hepatology. 26. 1092-1099 (1997)

[Publications] TAKEMURA Masaharu: "Phosphorylated Retinoblastoma Protein Stimulates DNA Polymerase α." Oncogene. 15. 2483-2492 (1997)

[Publications] MIZUSHINA Yoshiyuki: "The Inhibitory Action of Fatty Acids on DNA Polymerase β." Biochim.Biophys.Acta. 1336. 509-521 (1997)

[Publications] TAKEUCHI Eiji: "Research review : DNA polymerases as molecular markers of the regenerating capacity of hepatocytes." J.Hep.Bil.Pancer Surg.4. 254-262 (1997)

[Publications] HORIE Tadashi: "A5'-monophosphate Form of Bredinin Selectively Inhibits the Activities of Mammalian DNA Polymerases In Vitro." Int.J.Mol.Med.1. 83-90 (1998)

[Publications] KATO,Osamu: "Serum DNA Polymerase β as an Indicator for Fatal Liver lnjury of Rat Induced by D-Galactosamine Hydrochloride and Lipopolysaccharide." Biochim.Biophys.Acta. (in press).

[Publications] 吉田松年: "DNA複製・修復と発癌(松彰昭夫編)" 羊土社, (1996)

吉田松年名古屋大学, 医学部, 教授 (70090420)